1. Field of the Invention
The present invention relates to methods, compositions and kits for performing polyaromatic hydrocarbon (PAH) immunoassays.
2. Discussion of the Background
Testing is an essential, and integral, component of all environmental protection and restoration activities. It is the rate limiting element that influences the time, cost, and overall efficiency of project management.
The Environmental Protection Agency (EPA) has long promoted and supported the concept of screening methods to supplement laboratory analysis and increase overall efficiency. The need for more effective methods has been recognized in the Superfund Amendments and Reauthorization Act of 1986 which specifies the development and evaluation of alternative time and cost-saving methods that will assist in the eventual remediation of the nations Superfund sites.
Effective field screening methods can increase the efficiency of site management and improve overall data quality when used to supplement the services of regional laboratories. The development of these methods, however, requires a technology that will be compatible with numerous compounds and matrixes and yet be simple, effective and rugged enough to be incorporated into a protocol for use in the field.
Screening methods need to provide fast, simple, cost-effective and reliable information when operated under field conditions. The reagents and equipment should be portable and stable at ambient conditions, and the claims relating to performance should accurately reflect anticipated field use. The methods should be able to rapidly provide an ample quantity of data, and the protocol should be simple to perform and safe to use. Performance characteristics relative to sensitivity, freedom from matrix interferences and cross-reacting compounds, and correlation to an acceptable reference method should be carefully evaluated. Developers must maintain high, and consistent, quality standards relative to the consistency of their manufacturing protocols, the adequacy of in-process and pre-release quality control methods, and the reliability of their product claims. A necessary characteristic of particular significance for screening methods, is that they exhibit a very low frequency of false negative results.
Screening methods detect contamination at specified concentrations. The concentration may relate to a hazardous threshold, a clean-up target, or a process-control parameter. The potential implications of false negative data far outweigh those of false positive results. The consequence of a false positive, while a costly problem that needs to be minimized, results in additional testing or treatment. False negative data, however, provides an erroneous perception of a clean site, and may have serious environmental and legal consequences. Safeguards that minimize the incidence of false negative results are imperative. Appropriate control over the frequency of false positive data needs to be established and maintained.
Contamination of soil with polyaromatic hydrocarbons (PAH) is a serious problem at manufactured gas plant sites, coking operations, wood preserving operations that have used creosote as a wood preservative, and petrochemical plant waste disposal sites. The federal and state regulatory agencies are mandating the clean-up of many of these sites due to the carcinogenic nature of some of the PAHs.
Polyaromatic hydrocarbons (PAHs), or polynuclear aromatic compounds (PNAs), are fused ring aromatic compounds classified by the number of carbon rings. Sixteen of these PAHs are listed as hazardous compounds by the EPA (see Table I below).
TABLE I ______________________________________ Listed PAH Compounds ______________________________________ acenaphthene acenaphthylene anthracene benzo[a]anthracene benzo[a]pyrene benzo[b]fluoranthene benzo[g,h,i]perylene benzo[k]fluoranthene chrysene dibenzo[a,h]anthracene fluoranthene fluorene indeno[1,2,3-cd]pyrene naphthalene phenanthrene pyrene ______________________________________
They are further divided into carcinogenic and non-carcinogenic PAHs. The two and three ring PAHs are non-carcinogenic, while several of the four, five and six ring PAHs are carcinogenic. The four ring PAHs, chrysene and benzo[a]anthracene, the five ring PAHs, benzo[a]pyrene, benzo[b]fluoranthene, benzo[k]fluoranthene, and dibenzo[a,h]anthracene, and the six ring PAH, indeno[1,2,3-cd]pyrene, are listed as carcinogenic PAHs with benzo[a]pyrene being the most potent carcinogen among the PAHs. Regulatory concern is generally focused on benzo[a]pyrene, total carcinogenic PAHs, and total PAHs.
The laboratory methods usually employed for PAH analysis, EPA methods 8270 (GC/MS) and 8310 (HPLC), are relatively expensive, typically $200-500, and suffer from the same laboratory turnaround time requirements (2-4 weeks) as other lab methods. While the minimum detection levels for these methods can be quite low (10-1500 ppb), PAH containing samples are usually quite "dirty", routinely resulting in much higher levels of practical quantitation, typically on the order of 1-100 ppm. Due to the complicated chemical nature of coal tar or petroleum residues, PAHs are difficult to analyze accurately using the conventional methods. Although the results obtained using the standard EPA methods do not show gross differences between labs, serious discrepancies are seen with the carcinogenic PAHs when they occur at high concentrations.
Immunoassay methods combine the specific binding characteristics of an antibody molecule with a read-out system that is used to detect and quantify compounds. Current immunoassay technology benefits from the diversity of detection systems developed that use enzyme-catalyzed chromogenic reactions, radionuclides, chemiluminescence, fluorescence, fluorescence polarization and a variety of potentiometric and optical biosensor techniques. Improvements in the sensitivity achieved has necessitated the generation of new descriptive nomenclature for methods that can now detect "zeptomolar" (10.sup.-21, 600 molecules) concentrations
The U.S. Environmental Protection Agency has concluded that immunoassay technology provides an advantage over the traditional analytical methods (gas chromatography (GC), gas chromatography/mass spectrometry (GC/MS), high performance liquid chromatography (HPLC) by increasing the sample throughput at a lower cost (Van Emon et al. in Field Screening methods for Hazardous Wastes and Toxic Chemicals, p. 815-818 (1991)). The EPA SW-846 Organic Methods Work Group have approved draft immunoassay methods for inclusion into the next revision of the SW-846 methods. From this information it is obvious that newly developed reagents and immunoassays are becoming acceptable analytical tools for the environmental field.
While various immunoassay methods have been described which target specific individual PAH compounds, these methods require coupling the PAH compound to a DNA fragment (see Harris, U.S. Pat. No. 4,794,074 and Wallin et al Cancer Lett,, 22(2), 163-70 (1984)), using a monoclonal antibody for analysis of only a single PAH compound in a single test (see Dougherty, E. P. 0,242,589) or using a monoclonal antibody to detect specifically benzo(a)pyrene and its metabolites in urine or water (see Roda et al, Environ. Technol., 12(11), 1027-35 (1991 and Gomes et.al, Chem. Res. Toxicol., 3(4), 307-10 (1990).) However, no immunoassay methods are available for the detection of PAHs in a variety of matrices of various origin and overall compositions.
Thus, an immunoassay method is needed which will provide reliable accurate and fast results in the field for PAH contaminants, regardless of origin or exact composition. Such an assay would increase the efficiency of environmental site management activities such as characterization (mapping), remediation monitoring, and regulatory compliance.